DNA methylation serves an important role in determining gene expression states, and aberrant methylation and gene silencing contributes to the development of multiple tumor types, including glioblastomas (GBM). Similar to genetic alterations, substantial intratumoral heterogeneity in aberrant methylation at particular loci suggests there may be subpopulations of GBM cells defined in part by distinct epigenetic profiles. A subpopulation of primary GBM cells (CD133+) is capable of initiating tumor formation in nude mice, and thus appear to contain cancer stem cells. The CD133+ subpopulation is capable of producing tumorigenic and non-tumorigenic progenitors, suggesting that differences between subpopulations are likely epigenetic, however the extent to which aberrant methylation and gene silencing influences the CD133+ cells has not been addressed. Defining the differences in the epigenomes of each subpopulation would be a critical first step to addressing the hypothesis that aberrant CpG island methylation and gene silencing are necessary to endow CD133+ cells with cancer stem cell properties. The Costello laboratory has developed a novel technique to assess the methylation status of CpG islands on a genome-wide scale using existing BAG array technology. These arrays will be used to define the patterns of copy number change and aberrant CpG island methylation in the tumorigenic, CD133+, and the non-tumorigenic, CD133-, subpopulations of 30 surgically resected primary GBMs (Aim 1). Treatment with demethylating agents will determine if the aberrant CpG island methylation is sufficient to silence gene expression in primary tumors, and whether the effect is reversible in vitro (Aim 2). To determine if gene re-activation by demethylation is sufficient to reverse the tumorigenic potential of CD133+ cells or lack of potential of CD133- cells in vivo (Aim 3), nude mice will be injected with treated and untreated CD133 fractions and assessed for their ability to form tumors. The long-term goal of these experiments will be to study in-depth individual gene(s) that are methylated and silenced specifically in CD133+ or CD133- cells, reactivated in cells treated with the DNA demethylating agent, and whose reactivation correlates with a change in the tumorigenic capacity of these cancer stem cells. These experiments will significantly advance our understanding of the contribution of epigenetic mechanisms to GBM development, which has been a largely genetic view until recently. Since epigenetic changes are reversible these data could help to identify selective targets for epigenetic therapy. [unreadable] [unreadable] [unreadable]